Silver di-t-butyl phosphate, a useful reagent in the synthesis of phospholipids. Synthesis of mixed-acid phosphatidic acid and phosphatidyl glycerolphosphate

The synthesis of silver di-t-butyl phosphate is described. Using this reagent, mixed-acid phosphatidic acid with one unsaturated fatty acid could be prepared by means of a reaction with a 1,2-diacyl glycerol-3-iodohydrin. The blocking groups could be removed easily with dry hydrogen chloride at low temperatures. Phosphatidyl glycerolphosphate was prepared by means of a double condensation reaction between 1,3-diiodo-2-t-butyl glycerol and the silver salts of 1,2-diacyl glycerol-3-(benzyl)phosphate and of di-t-butyl phosphate. The protecting groups were released by anionic debenzylation and treatment with hydrogen chloride. Some properties and enzymic degradations of the synthesized compounds are discussed

Silica gel stimulates the hydrolysis of lecithin by phospholipase A

1. 1. Silica gel stimulates the hydrolysis of aqueous lecithin suspensions by phospholipase A. The activation is slightly greater than that caused by ether and takes place equally well in bulk suspensions of silica gel or on thin-layer Chromatographic plates prepared with silica. 2. 2. The hydrolyses of lecithin by phospholipase C and of triolein by lipase are not affected by silica under the reaction conditions employed. 3. 3. In light of these findings, it is advisable to employ an independent means of stopping phospholipase A reactions prior to product separation in chromatographic systems

The synthesis of 3-phosphatidyl-1′-glycerol

The synthesis of 1-oleoyl-2-palmitoyl glycerol-3-phosphoryl-1′-glycerol is described. By means of a reaction between a 1.2-diacyl-glycerol iodohydrin and the silver salt of 2.3-isopropylidene glycerol-1-(benzyl)-phosphate and removal of the protecting groups phosphatidyl glycerol was obtained in the same stereochemical configuration as the naturally occurring substance. Confirmation of the structure of the synthesized compound was achieved with the aid of different enzymes and analytical procedures

Synthesis and enzymic hydrolysis of an o-alanyl ester of phosphatidyl glycerol

A racemic 0-alanyl ester of phosphatidyl glycerol, containing one saturated and one unsaturated fatty acid, was synthesized by a reaction between silver benzyl- (γ-oleoyl-β-palmitoyl)-Dl-[alpha]-glycerol phosphate and DL-[alpha]-iodo-B-tert.-butyl-y-(N-tert.-butoxycarbonyl)-m-alanyl glycerol. The synthetic substance was hydrolysed by phospholipase A (EC 3.1.1.4), C (EC 3.1.4.3) and D. (EC 3.1.4.4). The results of the enzymic degradation and some other properties of this compound have been compared with those of amino acid derivatives of phosphatidyl glycerol from bacteria

Studies on cardiolipin III. Structural identity of ox-heart cardiolipin and synthetic diphosphatidyl glycerol

Chemical synthesis of diphosphatidyl glycerol, a long-chain fatty acid ester of diphosphatidyl glycerol, phosphatidyl diglyceride and phosphatidyl glycerophosphate has stimulated a structural comparison with natural cardiolipin. Although in certain properties the various polyglycerol phospholipids are quite similar, the results of enzymic hydrolyses with phospholipase A (EC 3.1.1.4), acylation studies, optical rotation measurements and chromatography of the intact phospholipids and deacylated products indicated that beef-heart cardiolipin has a diphosphatidyl glycerol structure. Conclusive evidence was obtained by means of the breakdown of the phospholipids with phospholipase C (EC 3.1.4.3). The enzyme was found to hydrolyse both natural and synthetic diphosphatidyl glycerol into 1,2-diglyceride and 1,3-glycerol diphosphate, phosphatidyl glycerophosphate being an intermediate hydrolysis product

Structural investigations on glucosaminyl phosphatidylglycerol from Bacillus megaterium

1. 1. Glucosaminyl phosphatidylglycerol from Bacillus megaterium was converted into phosphatidylglycerol and 2,5-anhydromannose. The stereochemical configuration of phosphatidylglycerol was investigated with phospholipase A, phospholipase C, and glycero-3-phosphate dehydrogenase, and was found to be 1,2-diacyl-sn-glycero-3-phosphoryl-1′-sn-glycerol. 2. 2. Partial acid hydrolysis of the phospholipid yielded glucosaminylglycerol, which was shown to be identical with 2′-O-(2-amino-2-deoxy-β-glucopyranosyl)-glycerol 3. I’-O-(z-Amino-z-deoxy-~-D-glucopyranosyl)glycerol, z’-O-(2amino-2-deoxy- I-D-glucopyranosyl)glycerol and z’-O-(z-amino-z-deoxy-~-D-glucopyranosyl)-I’-phosphorylglycerol were synthesized and compared with glucosaminylglycerol and glucosaminyl glycerophosphate derived from the phospholipid. 4. The structure of the phospholipid was established as r,z-diacyl-sn-glycero- 3-phosphoryl-I’[2’-0-(2R-amino-z)’-deoxy-D-glucopyranosyl)]-sn-glycerol

The synthesis of 3-phosphatidyl-1′-glycerol

The synthesis of 1-oleoyl-2-palmitoyl glycerol-3-phosphoryl-1′-glycerol is described. By means of a reaction between a 1.2-diacyl-glycerol iodohydrin and the silver salt of 2.3-isopropylidene glycerol-1-(benzyl)-phosphate and removal of the protecting groups phosphatidyl glycerol was obtained in the same stereochemical configuration as the naturally occurring substance. Confirmation of the structure of the synthesized compound was achieved with the aid of different enzymes and analytical procedures

Synthesis and enzymic hydrolysis of an o-alanyl ester of phosphatidyl glycerol

A racemic 0-alanyl ester of phosphatidyl glycerol, containing one saturated and one unsaturated fatty acid, was synthesized by a reaction between silver benzyl- (γ-oleoyl-β-palmitoyl)-Dl-[alpha]-glycerol phosphate and DL-[alpha]-iodo-B-tert.-butyl-y-(N-tert.-butoxycarbonyl)-m-alanyl glycerol. The synthetic substance was hydrolysed by phospholipase A (EC 3.1.1.4), C (EC 3.1.4.3) and D. (EC 3.1.4.4). The results of the enzymic degradation and some other properties of this compound have been compared with those of amino acid derivatives of phosphatidyl glycerol from bacteria

Studies on phospholipase A and its zymogen from porcine pancreas III. Action of the enzyme on short-chain lecithins

1. 1. Short-chain lecithins (with C6, C7, and C8 fatty acid esters) have been used to study kinetically the enzymatic hydrolysis by pancreatic phospholipase A (EC 3.1.1.4) in aqueous systems, without the addition of emulsifiers. 2. 2. Although phospholipase A is able to attack these substrates in molecularly dispersed form, micellar solutions are hydrolyzed at a much higher rate. 3. 3. Of the three substrates examined, dioctanoyllecithin appeared to be the best substrate. Differences in maximal velocities might be interpreted in terms of interfacial area per molecule. 4. 4. Ca2+ is specifically required for activity of pancreatic phospholipase A. The kinetic results are consistent with a random mechanism in which the metal ion combines with the enzyme independently of the substrate. The substrate was found to combine with the enzyme independently of the metal ion concentration. 5. 5. Kinetic parameters were determined with diheptanoyllecithin as a substrate over a pH range from 5 to 9. Maximal binding of enzyme with substrate was observed at pH : 6. The affinity of the enzyme for Ca2+ decreased at pH values below 6.5. 6. 6. With diheptanoyllecithin as substrate, maximal velocities at infinite substrate and Ca2+ concentrations showed an optimum at pH 5.75. 7. 7. NaCl at high concentrations (up to 3.9 M) gave a 80-fold stimulation of the vmax (diheptanoyllecithin as substrate). The Ks value decreased slightly with increasing salt concentrations, while the KCa2+ increased very strongly. The activating effect of salt is presumed to be caused by a change of the properties of the lipid-water interface

Studies on phospholipase a and its zymogen from porcine pancreas IV. The influence of chemical modification of the lecithin structure on substrate properties

1. 1. A series of chemically modified lecithins were used to investigate by kinetic analyses their substrate c.q. inhibitor properties for porcine pancreatic phospholipase A. The substrate analogues used were systematically modified in: the stereochemical configuration, the susceptible ester bond, the phosphate moiety, the alkylchains, the glycerol backbone and in the position of the phosphorylcholine moiety. 2. 2. The desired relationship between chemical structure and inhibitory properties requires elimination of purely physical effects of the inhibitor on the organization of the substrate molecules at the lipid-water interface. 3. 3. Lecithins of the opposite stereochemical configuration and certain lecithin analogues with a modification of the susceptible ester bond were found to be purely competitive inhibitors. The 1-sn-phosphatidylcholines have Ki values identical to the K8 values of the corresponding 3-sn-phosphatidylcholines. The lecithin analogues with an acylamide linkage at the 2-position were found to be the most potent competitive inhibitors, while on the contrary substitution of the acylester bond by a sulfonyl ester linkage does not give rise to inhibitory properties. 4. 4. Lecithins with a modification in the glycerol-phosphate bond and in the position of the phosphorylcholine moiety are substrates, but exhibit much lower V values and their binding constants are similar to those of the corresponding normal substrates. 5. 5. Introduction of two methyl groups at the carbon atom adjacent to the carboxyl in the acyl chain of the potentially susceptible ester bond gives a lecithin which is not degraded by the enzyme. The presence of only one methyl branch in this position greatly diminishes the hydrolysis rate, probably due to steric hindrance. 6. 6. Increasing the distance between the susceptible ester bond and the phosphate moiety in a lecithin by introducing a methylene group completely abolishes enzymatic activity. These lecithin analogues were found to be competitive inhibitors. 7. 7. The minimal requirements for a phospholipid to be a substrate for phospholipase A, as established earlier, should be extended to include the fact that the phosphate moiety can be replaced by a phosphonate or sulfonate group